Enzymatic hydrolysis of geniposide from gardenia jaminoides to produce genipin as a pigment precursor and crosslinking agent

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION THESIS FOOD TECHNOLOGY ENZYMATIC HYDROLYSIS OF GENIPOSIDE FROM GARDENIA JAMINOIDE TO PRODUCE GENIPIN AS A PIGMENT PRECURSOR AND CROSSLINKING AGENT SUPERVISOR: VO THI NHA NGUYEN VINH TIEN STUDENT: DANG HOANG DUC HO DAC LOC SKL 0 Ho Chi Minh City, August, 2022 HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION PROJECT Thesis code 2022-18116009 ENZYMATIC HYDROLYSIS OF GENIPOSIDE FROM GARDENIA JAMINOIDE TO PRODUCE GENIPIN AS A PIGMENT PRECURSOR AND CROSSLINKING AGENT DANG HOANG DUC Student ID: 18116009 HO DAC LOC 18116022 Major: FOOD TECHNOLOGY Supervisor: VO THI NGA, PhD NGUYEN VINH TIEN, ASSOC PROF Ho Chi Minh City, August 2022 HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION PROJECT Thesis code 2022-18116009 ENZYMATIC HYDROLYSIS OF GENIPOSIDE FROM GARDENIA JAMINOIDES TO PRODUCE GENIPIN AS A PIGMENT PRECURSOR AND CROSSLINKING AGENT DANG HOANG DUC Student ID: 18116009 HO DAC LOC 18116022 Major: FOOD TECHNOLOGY Supervisor: VO THI NGA, PhD NGUYEN VINH TIEN, ASSOC PROF Ho Chi Minh City, August 2022 DECLARATION Except where there is clear acknowledgment and reference to the work of others, we thus declare that all content and materials included in and presented in this thesis are our original creations Additionally, we guarantee that the materials acknowledged in the thesis have been cited appropriately and properly in line with requirements , August 2022 Signature I ACKNOWLEDGEMENT "No research without action, no action without research" is a well-known quote attributed to Kurt Lewin (2012) Indeed, in order to finish and attain the current outcome, we have addressed and conquered all the difficulties and obstacles inherent in and throughout the project In addition, our beloved professors, classmates, and families have made a substantial contribution that stimulates and supports us Therefore, we would like to sincerely thank all the lecturers in charge of the Department of Food Technology, Faculty of Chemical and Food Technology, and Ho Chi Minh City University of Technology and Education for providing us with valuable knowledge and the best equipment and facilities to complete our thesis We would also want to express our gratitude to our cherished supervisors, PhD Vo Thi Nga and Assoc Prof Nguyen Vinh Tien, who have passionately guided and shared their teaching expertise and experience in order for us to complete this thesis Sincerely, we would like to thank Ms Ho Thi Thu Trang of the Department of Food Technology for allowing and assisting us in using the available measuring instruments and equipment at the Faculty of Chemical and Food Technology laboratory Nonetheless, we would want to express our gratitude to our classmates for supporting and organizing the thesis's complex experiment II III IV V VI genipin, varying from 1528 cm-1 to 1512 cm-1 at pH 12 It is possible that beans are very soluble in pH 12, and when reacting with genipin, the amine of the bean protein reacts with genipin, so we can see that the amine peak there varies markedly between samples These structural changes could result from electrostatic interactions between the protein's amine groups And we can see that the peak of the secondary amine does not change significantly The O-H stretching vibration is visible in the FT-IR pattern as a sharp peak for the control protein sample at wave number 3266 cm-1, which shifts to wave numbers 3278 cm-1 and 3274 cm-1 for the samples, respectively, pH 10 and pH 11 Additionally, for the pH 10 and pH 11 samples, the FT-IR sample shape widened and altered Furthermore, compared to the control sample, the pH sample had a significantly larger diameter with the same wave number, 3266 cm-1 This positional shift may result from creating hydrogen bonds between the protein and genipin chains.[68] A peak may be seen at wavenumber 2925 cm-1, and it is caused by complex oscillating stretching involving both the free and inter-molecular CH group [70] However, at this peak, we detect no difference between samples 3.3 Properties of chitosan-genipin films After investigating all the factors affecting the blue color production from genipin We continue to investigate the properties of the genipin - Chitosan film to see the quality of the film 3.3.1 Uv-vis of films when change genipin content 46 Fig 22 UV-vis spectra of genipin-chitosan films This experiment shows changes in crosslinking between samples that change the genipin concentration This experiment is evaluated according to absorbance There were five samples changed according to the increasing concentration of genipin, one sample was completely chitosan, and the remaining four samples were in order of increasing genipin concentration from 0.0025 to 0.01 The absorption spectra of films based on chitosan crosslinked with genipin at various genipin/chitosan ratios are shown in Figure 3.22 The absorption spectra of pure chitosan show weak bands below 400nm Films containing genipin exhibit intense absorption bands in the 260-700 nm range [57] The results show that at a wavelength of about 600–610 nm, the absorbance reaches its maximum; we can see that the sample of 0.01 genipin/NH2 has the highest absorbance compared to the remaining samples and tends to decrease gradually when reducing the concentrations genipin in the sample The reaction of radical polymerization of genipin, caused by oxygen in the air, which takes place as one of the stages in the process of crosslinking chitosan, was attributed to the absorption band at 610 nm [71] And also, at the wavelength range of 280-290 nm, the absorbance of the samples also peaked; we can see that the 47 trend is quite similar to the absorption at about 600-610 nm The inclusion of chitosan amino group in the heterocycle of genipin, which results in the formation of heterocyclic amine, was related to the absorption band at 280-290 nm [71] The rise in peak intensity between 280 and 290 nm shows a greater degree of genipin binding by chitosan at a high genipin/NH2 ratio [57] 3.3.2 FTIR of films when change genipin content Fig 23 FTIR of genipin-chitosan films After letting the film dry, we conduct FTIR measurement to know how the structure of chitosan will change when acting with genipin In figure 3.23 there are samples with varying concentrations from 0.0025 to 0.01 genipin/NH2 and a control chitosan sample The spectrum of chitosan is characterized by a number of absorption bands; the peaks at 1653 cm-1 and 1549 cm-1 were attributed to N-H bending in amide II and C=O stretching in amide I, respectively The unique asymmetric stretching of the C-O-C polysaccharide structure was attributed to the band at 48 1155 cm-1 [72] Following other FTIR studies on genipin treated biopolymers , the samples showed additional peaks after genipin cross-linking at 1281, 1423, and 1639 cm-1 in 0.0025 genipin/NH2 These peaks were attributed to the C-O-C asymmetric stretching, the CH3 bending of the methyl ester, and the C=C ring stretching, respectively According to the literature [73], these characteristics imply that the carboxymethyl group of genipin reacted with the amino group of chitosan to generate a secondary amide From the results of Figure 3.22, we can see that when increasing the concentration of genipin in the chitosan solution, the peak will change with an increasing trend from 1638 to 1653 cm-1 , which means that the FTIR spectrum tends to be wider At the peak of 1281 cm-1, there is also an increasing trend But at the peak of 1423cm-1, there is a decreasing trend The C-N stretch of the tertiary aromatic amine of the cross-linked genipin nitrogen iridoid, which is covalently attached to the chitosan, was identified as the source of the band at 1112 cm-1 [73] Additionally, the cross-linked spectrum's band at 1070 cm-1 is noticeably stronger than the comparable band in the spectrum of pure genipin, indicating that this absorption band is primarily connected to modes created by the cross-linking process From the results, the peak 1070 tends to decrease when increasing genipin concentration, this can be understood that when increasing genipin concentration, the ability to cross-link is higher 3.3.3 Moisture content of genipin – chitosan films (%) Table Moisture content of genipin – chitosan films genipin/ NH2 content Moisture content (%) 9.21±0.006d 0.0025 7.87±0.007c 0.005 5.41±0.009b 0.0075 3.47±0.004a 0.01 2.01±0.007a Note: different lowercase letters in a column illustrate significant differences (p < 0.05) The purpose of measuring the moisture content of the film is to see the change in moisture content when genipin cross-links with chitosan From the results of Table 3.6, we can see that the moisture content of the samples compared with the control samples gradually decreased 49 Includes four samples according to increasing genipin concentration in chitosan solution from 0.0025 to 0.01 genipin/NH2 Perhaps it was the reaction between genipin and chitosan that changed the structure of genipin Specifically, genipin will have membranes that block water molecules that can easily penetrate 3.3.4 Mechanical properties of genipin – chitosan films (Thickness, TS and EL) Table Mechanical properties of genipin-chitosan films Genipin/ NH2 content Thicknes(mm) Tensile strength(N/mm2) Elongation (%) 0.104±0.013a 21.77±6.56a 227.12±14.06b 0.0025 0.098±0.003a 18.42±0.99a 161.72±79.8a 0.005 0.107±0.010a 16.43±1.19a 163.22±18.77a 0.0075 0.106±0.007a 23.46±2.75a 179.72±0.48a 0.01 0.099±0.001a 19.92±3.50a 182.64±5.12a Note: different lowercase letters in a column illustrate significant differences (p < 0.05) The goal of this experiment is to measure the tensile strength and elongation of the films, as well as their flexibility and mechanical resistance, which determine the film's integrity and sustainability criteria when change genipin concentrations To obtain a film that is stable and insoluble in water, we must find the concentration of genipin to put in chitosan Here we have investigated concentrations of genipin and a control sample without genipin The largest sample was 0.01 genipin/NH2 and the smallest sample was 0.0025 genipin/NH2 The amount of crosslinking reagent required to complete the crosslinking reaction is less than that needed for gel formation in a 2% chitosan solution, and the concentration of the solution rises as a result of film formation during solvent evaporation The film created at this genipin/NH2 ratio was colorless for 24 hours following solvent evaporation before becoming blue and water-repellent [57] However, reducing the genipin content in the chitosan solution not only did not reduce the strength but even increased the strength of the film As a result of Table 3.7, we can see that the films with a concentration of 0.0075 genipin/NH2 has higher durability than the film with a concentration of 0.01 genipin/NH2 because the crosslinking of the chitosan solution was not fully 50 completed before the solvent evaporated the macromolecular chains of chitosan and may also result from the chains' immobilization in the equilibrium state, where more intermolecular interactions take place [57] 3.3.5 Swelling content of genipin – chitosan films Table Swelling content of genipin – chitosan films Genipin/ NH2 content Swelling content (%) 165.17±10.27c 0.0025 148.41±21.47b 0.005 129.69±3.70a 0.0075 121.31±1.79bc 0.01 109.62±4.04d Note: different lowercase letters in a column illustrate significant differences (p < 0.05) In this experiment, we can see that the water repellency of the film is based on different genipin concentrations In table 3.8, we can see that the swelling ratio of 0.0025 genipin/chitosan film is the highest and tends to decrease with low genipin concentration The swelling ratios of the crosslinked chitosan films were reduced as the concentration of genipin utilized was increased [74] Perhaps because the crosslinking between genipin and chitosan prevented water from entering and deepening the internal structure, when the genipin content was increased, the crosslinking became denser Therefore, it is harder for water molecules to penetrate 51 CHAPTER 4: CONCLUSION From the experimental results, some conclusions are drawn as follows: - A method for preparing gardenia blue pigment that includes the following steps: a) Geniposide is subjected to cellulase to produce a hydrolysate b) The hydrolysate produced in step a) is extracted with EtOAc and the solvent is removed after extraction to provide a genipin-containing product c) The genipin-containing product from step b) is dissolved in ethanol and then reacted with an aqueous solution of an amino acid or its salt to form the gardenia blue pigment In roughly 0.2 g of cellulase per g of geniposide, cellulase is introduced to the reaction in step a) - The optimal conditions of the study: The pH of the treatment performed in step a) is 4.5 The duration of the treatment performed in step a) is completed in hours Monosodium glutamate is the amino acid that is chosen to react with genipin in step c) to produce the gardenia blue pigment The pH of the treatment performed in step a) is The duration of the treatment performed in step a) is completed in 10 hours - For the process of reacting between genipin and protein extracted from Lima bean: the most preferable pH for the reaction between genipin and protein extracted from Lima bean is 10 - When genipin reacts with amino acids, amino acids give the best results specifically, the solution is blue pigment And when genipin with primary and secondary amines, there is still a pigment forming reaction but it is not blue - When changing the concentration of genipin/NH2 from small to large, the cross-linked genipin-chitosan film and the blue pigment increased In terms of mechanical properties, with a 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